Abstract # A-08

Separation of a Newly-Identified Contaminant from Commercial 4,5,6,7-Tetrachlorofluorescein by pH-Zone-Refining Countercurrent Chromatography. A. Weisza, D. Andrzejewskib, R. J. Highetc, and Y. Itoc.  aOffice of Cosmetics and Colors and bOffice of Scientific Analysis and Support, US Food and Drug Administration, Washington, DC 20204, USA, cLaboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA

The compound 4,5,6,7-tetrachlorofluorescein (TCF) is an intermediate in the preparation of the U.S.- certified color additives D&C Red No. 27 (mainly 2',4',5',7 -tetrabromo-4,5,6,7-tetrachlorofluorescein) and its disodium salt, D&C Red No. 28 (Phloxine B). Commercial TCF was found to contain many impurities, several of which were recently separated by pH-zone-refining countercurrent chromatography (CCC). The impurities can be carried over to the color additives during the manufacturing process, thus reducing the probability of their complying with FDA certification specifications. In the present study, pH-zone-refining CCC was successfully applied to the separation of a less polar isomer of TCF that was present in all batches of TCF obtained from several different suppliers. The isolated material was identified by mass spectral and NMR analyses. The difference between the mass spectral behavior of TCF and that of its isomer may be of diagnostic importance in distinguishing these two compounds.